PROJECT SUMMARY/ABSTRACT Toll-like receptor (TLR) signaling plays a crucial role in mediating innate immunity and, when deregulated, also contributes to the pathogenesis of inflammatory diseases. Better understanding of the molecular mechanisms regulating TLR signaling and inflammatory responses is highly significant for improving the therapeutic approaches in the treatment of inflammatory diseases. During the previous funding cycles, the PI?s laboratory has made seminal discoveries in this area. Moreover, we have generated a large body of innovative preliminary data that form a solid foundation for this continuation application. In particular, our preliminary studies demonstrated a crucial role for the protein kinase, TBK1, in controlling TLR signaling and preventing inflammatory disorders. Although TBK1 is known as a kinase that mediates type I interferon (IFN) induction and antiviral innate immunity, its in vivo functions have been poorly studied due to the lack of a viable mouse model. Using newly generated TBK1 conditional knockout (cKO) mice, we have discovered novel functions of TBK1 in the regulation of immune and inflammatory responses. Our preliminary studies have demonstrated a crucial role for TBK1 in controlling inflammatory responses by functioning in both innate immune cells and intestinal epithelial cells (IECs). Myeloid cell-conditional TBK1 KO (Tbk1-MKO) mice are hypersensitive to colitis induction and spontaneously develop aberrant adipose tissue expansion and inflammation. TBK1 negatively regulates TLR signaling and TLR-stimulated expression of proinflammatory cytokines in macrophages. We have further demonstrated that conditional deletion of TBK1 in IECs increases proinflammatory cytokine production and Th17 cell generation in the intestine, sensitizing mice for intestinal tumorigenesis. Based on these innovative findings, we hypothesize that TBK1 functions in both innate immune cells and IECs to regulate proinflammatory TLR signaling and inflammatory disorders. The overall objective of this continuation application is to elucidate the mechanism underlying the anti-inflammatory functions of TBK1. To accomplish this overall objective, we will perform two specific aims. In Aim 1, we will examine how myeloid cell TBK1 regulates TLR signaling and inflammation. In Aim 2, we will elucidate the mechanism by which TBK1 functions in IECs to regulate intestinal immune homeostasis and tumorigenesis. We believe that these proposed studies address novel mechanisms that regulate TLR signaling and inflammatory responses and will lead to high-impact results that substantially advance the field.